1-imino-1H-isoindol-3-amine

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.12.2016-01.03.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-imino-1H-isoindol-3-amine

EC Number:

222-426-8

EC Name:

1-imino-1H-isoindol-3-amine

Cas Number:

3468-11-9

Molecular formula:

C8H7N3

IUPAC Name:

1-imino-1H-isoindol-3-amine

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
- Pre-incubation: the inserts containing the tissues were set into the incubator at 37 ± 1°C, 5.0% CO2 / 95% air for 1 hour.
- For each experiment (“3 minutes” and “1 hour”), the plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2.
- MTT medium: the tissues were incubated with MTT medium for 3 h at 37 ± 1°C, 5.0% CO2 / 95% air.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg test item + 25 µL H2O

Duration of treatment / exposure:

3 and 60 minutes treatment

Number of replicates:

2 replicates for each treatment period (3 min and 60 min exposure time)

Test system

Details on study design:

Details of the test procedure
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (MatTek, EPI-200-SCT)
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: the tissue was gently rinsed about 20 times with PBS
- Number of tissue replicates used per test chemical and controls: 2
- MTT assay: incubation with 0.3 mL of MTT solution for 3 hours at 37 ± 1 °C, 5% CO2)

- Data evaluation:
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%) according to the following Prediction Model
In step 1:
< 50% after 3 min exposure: predicted as corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure: predicted as corrosive (a combin
ation of optional sub-categories 1B and 1C)
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: predicted as Non-Corrosive
In step 2:
< 25% after 3 min exposure: optional Sub-category 1A
≥ 25% after 3 min exposure: a combination of optional Sub-categories 1B and 1C

- Historical data negative control: Mean Absorption: 1.895 (3 min), 1.867 (1 h); Standard Deviation: 0.313 and 0.261, respectively
- Historical data positive control: Mean Viability: 6.1% (1 h); Standard Deviation: 1.99%

- The test meets acceptance criteria if:
- mean OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min. incubation time

Value:

>= 87.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive to skin

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour incubation time

Value:

>= 17.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: borderline result

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour incubation time

Value:

>= 20.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: repeated due to a borderline result

Remarks:

no corrosive effects

Other effects / acceptance of results:

The controls confirmed the validity of the study. The mean OD570 of the two negative control tissues was≥ 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (5.4% and 10.7%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (2.8% - 13.3%).

Any other information on results incl. tables

Results of the 3 min experiment:

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	2.006	1.759	1.723	1.516	0.098	0.180
	2.021	1.738	1.839	1.490	0.097	0.182
	2.055	1.751	1.883	1.525	0.099	0.189
OD570- Blank Corrected	1.963	1.715	1.680	1.473	0.055	0.137
	1.977	1.695	1.796	1.447	0.054	0.139
	2.011	1.708	1.840	1.482	0.056	0.146
Mean OD570of 3 Aliquots (blank corrected)	1.984	1.706	1.772	1.467	0.055	0.141
SD OD570 of 3 Aliquots	0.025	0.026	0.078	0.029	0.024	0.024
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.845		1.619		0.098
TODTT	-		1.608		-
SD OD570 of 2 Replicate Tissues	0.197		0.215		0.061
Mean Relative Tissue Viability [%]	100.0		87.8		5.3
NSMTT-corrected mean relative tissue viability [%]	-		87.2		-
Coefficient Of Variation [%]	10.7		13.3		62.2

Results of the 60 min experiment:

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.627	1.683	0.346	0.275	0.155	0.110
	1.601	1.733	0.339	0.278	0.155	0.111
	1.641	1.721	0.339	0.270	0.156	0.113
OD570- Blank Corrected	1.582	1.638	0.302	0.230	0.110	0.065
	1.556	1.688	0.294	0.233	0.110	0.066
	1.596	1.676	0.294	0.226	0.111	0.068
Mean OD570of 3 Aliquots (blank corrected)	1.578	1.667	0.296	0.230	0.111	0.066
SD OD570 of 3 Aliquots	0.020	0.034	0.025	0.025	0.025	0.025
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.623		0.263		0.088
TODTT	-		0.289		-
SD OD570 of 2 Replicate Tissues	0.063		0.047		0.031
Mean Relative Tissue Viability [%]	100.0		16.2		5.4
NSMTT-corrected mean relative tissue viability [%]	-		17.4		-
Coefficient Of Variation [%]	3.9		17.9		35.5

Because of the borderline result, a second experiment was conducted.

Results of the second 60 min experiment:

Name	Negative Control		Test Item		Positive Control
Tissue	1	2	1	2	1	2
Absolute OD570	1.644	1.707	0.329	0.393	0.209	0.229
	1.700	1.672	0.352	0.400	0.213	0.228
	1.826	1.598	0.351	0.383	0.213	0.232
OD570- Blank Corrected	1.601	1.664	0.285	0.349	0.166	0.185
	1.657	1.628	0.309	0.357	0.169	0.184
	1.783	1.554	0.308	0.340	0.169	0.189
Mean OD570of 3 Aliquots (blank corrected)	1.680	1.616	0.301	0.349	0.168	0.186
SD OD570 of 3 Aliquots	0.093	0.055	0.027	0.025	0.024	0.024
Total Mean OD570of 2 Replicate Tissues (Blank Corrected)	1.648		0.325		0.177
TODTT	-		0.351		-
SD OD570 of 2 Replicate Tissues	0.046		0.034		0.013
Mean Relative Tissue Viability [%]	100.0		19.7		10.7
NSMTT-corrected mean relative tissue viability [%]	-		20.9		-
Coefficient Of Variation [%]	2.8		10.4		7.2

Applicant's summary and conclusion

Conclusions:

The test item showed no corrosive effects. The test item is classified as “non-corrosive“.

Executive summary:

In an in vitro study, the skin corrosion potential of the test item was assessed with the Reconstructed Human Epidermis (RhE)

test method according to OECD 431 and EU-Method B.40 and in compliance to GLP.

The test item was applied topically and cytotoxic effects to the stratum corneum after a short time exposure were determined. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

The test item showed non-specific MTT-reducing (NSMTT) potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT. After 3 min treatment, NSMTT was 0.6%, after 60 min treatment, NSMTT was -1.2%. The results were corrected accordingly. Moreover, the test item showed no colouring potential after mixture with aqua dest. but colouring was detected after mixture with isopropanol. Since there was no relevant absorption in the range of 570 ± 30 nm, NSC_living(non-specific colour of additional viable tissues) was not determined.

The corrosivity potential was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%). According to the Prediction Model: a mean relative tissue viability of ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure conclude to a non-corrosive effect of the test item. Due to a borderline result (17.4%) the 60 min treatment period was repeated. The mean relative tissues viability (% of negative control) was 87.2 % (NSMTT-redcuced) after 3 min treatment and was 17.4 % and 20.9% after 60 min treatment.

According to the Prediction Model, the test item showed no corrosive effects.